FcgammaRIII is a multisubunit complex (i.e. a ligand recognition subunit in association with disulfide-linked dimers composed of the signal transducing subunits zeta and gamma expressed on the surface of natural killer (NK) cells that is responsible for the recognition and elimination of antibody coated target cells. Whereas ligation of FcgammaRIII on the surface of NK cells is a potent activating stimulus, NK cells primed with IL-2 are induced to die in response to this same stimulus. We have found that IL-2 priming induces the expression of a CD95 (Fas)-zeta heterodimer, suggesting a molecular mechanism for activation-induced NK cell death. We will test the hypothesis that CD95 (Fas)-zeta is required for the activation-induced death of NK cells, and determine whether this phenomenon contributes to the selective elimination of zeta from tumor- infiltrating NK cells derived from patients with cancer. The specific aims are: i) To characterize CD95 (Fas)-zeta biochemically, ii) To define the role of CD95 (Fas)-zeta in activation-induced NK cell death, and iii) To determine how CD95 (Fas)-zeta signals apoptotic cell death. These aims will be accomplished by first determining the relative ability of zeta and gamma to associate with CD95 (Fas) in COS transformants, and using mutational analysis to define the dimerization domains. We will then identify activating stimuli capable of inducing the formation of CD95 (Fas)-zeta in NK cells, determine whether CD95 (Fas)-zeta associates with the FcgammaRIII complex, and assess the role of CD95 (Fas)-zeta in activation-induced NK cell death. Finally, we will measure the ability of CD95 (Fas)-zeta to induce tyrosine phosphorylation of zeta and to activate intracellular kinases involved in signaling apoptotic cell death. Identification of the molecular mechanisms responsible for activation-induced NK cell death might explain the depletion of NK cells expressing receptors coupled to zeta in cancer patients, which might in turn lead to the development of therapies aimed at preventing tumor- induced immunosuppresion.